This invention relates to a system, apparatus and method for processing samples and more particularly to performing (1) analytical procedures on samples based on complexation reaction principles and (2) affinity separations. Encompassed within this category of analytical procedures are tests based on immunoassay principles, nucleic acid hybridization principles, and affinity complexing principles. The complexation technology is applicable to a wide range of analytical applications including the detection of soluble antigens, cells, cellular fragments, microorganisms and their products. Encompassed within the category of affinity separations are the separations of biomolecules from complex mixtures.
Generally, the types of tests within this category of analytical procedures are procedurally complex, frequently they involve multiple reagent additions, extensive wash steps, and prolonged incubation times. These procedural complexities diminish the testing convenience. Furthermore, such tests must afford high sensitivity. In order to achieve this sensitivity, long reagent equilibration times are required in order to capture the minute quantities of analytes in samples.
Analytically, complexation based tests can be applied to a wide variety of analytes which are of diagnostic importance to clinical medicine and of investigative importance to life science research. The complexation tests are inherently sensitive and specific. However, as noted above, such tests can be labor intensive and require hours of analysis time to perform.
For example, a typical test can comprise the following generalized procedural steps. In the first step, sample fluids containing the analyte for testing is contacted with a solid phase "capture reagent"--prepared with a complexing reagent specific for the analyte of interest. The sample fluid is then equilibrated with the solid phase reagent for sufficient time to enable analyte attachment to the support. Following equilibration with the capture reagent, the sample fluid is removed and the solid phase reagent rinsed to remove excess sample materials. During this process the analyte attached to the capture support remains fixed on the surface of the capture support.
As a general rule for analytical purposes the attached analyte cannot be detected directly. A second "amplifying complexing reagent" often must therefore be added and equilibrated with the solid phase support in order to detect or visualize the presence of analyte. This equilibration must also be carried out for sufficient time to insure effective reaction with the analyte on the solid phase surface. Following equilibration, the excess "free" amplifying reagent must be separated from the "bound" amplifying reagent. This is accomplished by washing the solid phase support to eliminate the unfixed active elements. At this stage, depending on the nature of the amplifying reagent a direct reading of the active elements on the solid phase support can be taken. If a test is preferred using an enzyme or other tracer to amplify the test signal, one or more substrate reagents must first be added. Following an additional incubation period, color development can then be observed.
Unfortunately, the numerous manipulations required in performing the tests--notably during the successive reagent additions, rinsings, and incubations entail risk of errors in timing, reagent measurement, specimen identification, risk of user infection, and accidental loss of test samples. This is particularly true when the large numbers of samples are batched together at the same time. The problems encountered by diagnostic laboratories in carrying out these types of tests are thus many. Clinical laboratories must cope with large throughput of samples, interpret the significance of the results, provide a wide range of determinations, return results quickly, and ensure that each assay test is performed accurately. This must be done economically in spite of the difficulties encountered with techniques that are labor intensive, and complex when compared with other tests performed in clinical laboratory. (British Medical Bulletin, 30, 38-43, 1974).
While there are many variations by which such tests can be performed, a review of the practical and theoretical constraints are well documented and beyond the scope of this invention. To overcome these difficulties, numerous devices and automation approaches have been described in the prior art.
Currently, there are a number of automated machines which are commercially available. These types of machines make use of multi-well consumables such as microtiter plates or variations thereof. In general, these machines are useful in tests where quantitative information on analyte concentrations are required. Generally, these machines are complex and expensive.
To accommodate all of the testing functions mentioned above, the instrument designs make use of a multiple modules such as a sample application module, wash module, and plate reader or detector. Frequently, these systems are not fully automated requiring operator involvement to move the multi-well plate from one module to the next.
The most serious drawback to these types of systems results from their prolonged testing time and limited range of analytical application. Tests can require hours to days to perform. Prolonged equilibration times are requirred since the microtiter test wells provide relatively small surface area--consequently limiting the amount of complexing reagent available for reaction with the analyte. Furthermore, these systems tend to rely on simple diffusion or mechanical vibration to bring the analyte in contact with the surface of the test wells. As a consequence, equilibration generally require hours to accomplish. Test results are therefore not readily available in case of medical emergency or during the patient/physician interview.
A further limitation of these system is that they tend to be limited to immunoassay applications and not really adaptable to the newer complexation test based on DNA and RNA hybridization principles.
In order to overcome these limitations, a number of processor systems which make use of alternate test devices have been recently reported in the patent literature. For example, U.S. Pat. No. 4,071,315 issued Jan. 31, 1978 to G. Chateau discloses a processor concept which makes use of a complexing capture reagent attached to roll of porous film which is fed sequentially through a series of operational modules.
The Chateau system suffers from many of the same disadvantages as mentioned above. The system is complex depending on the function of multiple independent modules and relies on simple diffusion to effect mixing and accomplish analyte equilibration with the film reagent. A further disadvantage of this system is that the system does not provide rapid turnaround time for test results. However, once engaged, large numbers of samples can be run with high throughput.
U.S. Pat. No. 4,225,558 issued Sept. 30, 1980 to Peterson et al. describes a centrifugal technique in which a plurality of fluid test cells arranged on the periphery of a motor driven rotor. The fluids to be tested and respective reagents are introduced separately into corresponding test cells and are subsequently mixed in a reaction chamber for analysis. Introduction of the fluids is accomplished by the use of vacuum and the fluids are mixed by centrifugal force. This system has a disadvantage of requiring the use of centrifugal force which reduces the throughput of the system and renders the system unnecessarily complicated.
Another system is described in U.S. Pat. No. 4,424,279 issued Jan. 3, 1984 to Bohn et al. and U.S. Pat. No. 4,458,020 issued July 3, 1984 to Bohn et al. These patents, both assigned to Quidel, describe an apparatus for processing a cylindrical tube having an open end into which a plunger filter assembly is fitted. Beads sensitized with complexing reagent such as an antibody can be used in conjunction with the device. The operation is centered around a plunger which is depressed to mix the sample with the sensitized beads. Thereafter the reagent is added and the plunger is raised to clear the chamber of the fluids. The beads are washed in much the same way by raising and lowering the plunger. Although the system is simple in design, it suffers from the requirement that the reagents and wash fluids be added manually and that a four chambered dispensing unit and the filter tube assembly be manually moved from one position to the next in order to accomplish the assay. Operator involvement is extensive. Furthermore since the tube and filter assembly remain open during handling, the devices are subject to spillage and thus subject the user to potential contact with infectious materials.
Michael Cais and Moshue Shimoni, in Ann. Clin. Biochem. 18, 324-329 (1981) describes a tube device for performing immunoassays in which separation of "free" and "bound" analyte is claimed to be rapidly and safely accomplished by liquid extraction techniques. While it is not explicitly described they indicate that a simple automated instrument has been developed which processes up to 40 assay tubes simultaneously This device suffers from the disadvantage of being complex in design requiring high precision parts, and is limited in application to only those analytes which can be separated by solvent partitioning.
A device for separating plasma from a centrifugal blood sample is described in U.S. Pat. No. 4,483,825 issued Nov. 20, 1984 to Fatcher. This device includes a pipette having a filter disposed over one of the two open ends. The filter end is inserted into the tube holding the blood sample and operated like a piston to force the plasma through the filter into the pipette. Such device has not been used for complexation type testing. Furthermore, being open at both ends, the device would expose the operator to biohazardous materials.
In these prior art systems, auxiliary apparatus and equipment have been typically employed for the sequential exposure, equilibration and washing of the solid phase capture reagent. For example, a vibrator or shaker is useful to both maintain controlled uniform exposure of the reaction fluids with the capture reagent and to hasten the rate of analyte interaction with the support. In addition a centrifuge is useful and effective in the aggregation of suspended solid phase reagents following equilibration. An aspirator can then be used to facilitate the decantation of the reaction fluids from solid phase reagents. Peristaltic pumps or automated syringes are useful to add reagents and/or wash solutions. These many separate functions have not readily lent themselves to an uncomplicated single automation concept, at least in a single instrument.
A number of systems have been reported which make use of chromatographic principles to increase the efficiency of analyte capture and thus shortening the analysis time. German Patent Application No. DE3217-032-A describes an immunoassay separation process using dry chromatographic column materials. In this type of application, no automated system is described.
Other systems have been developed including those using manifold systems such as those offered by J. T. Baker and Waters, Inc. These systems are designed to speed up the flow and collection of column eluants by the use of vacuum. As such, these systems depend upon open columns in which fluid flows in only one direction. The processor consists simply of a vacuum source to suck the wash solution through the immunoreagents support. As a result, the test requires multiple tubes and considerable operator intervention to complete the test procedure.
Many flow-through type systems making use of a chromatographic type column bed for analyte capture and processing are well known in the immunoassay and bioseparation literature. However, the flow-through systems have the disadvantage that they make use columns which must be carefully packed in order to avoid both fluid channeling and the inclusion of trapped gas which may reduce fluid contact with the supports. This adds to the difficulty and cost to the manufacture of the column device. Furthermore during the process, analyte capture must be accomplished in a single pass necessitating a highly efficient column. This adds back pressure to the flow system frequently necessitating positive displacement pumps to compensate increased back pressure and insure positive fluid flow. Furthermore the simultaneous processing of multiple samples is not possible. In addition, during consecutive cycles, these systems are subject to contamination and plugging due to entrapment of particles and debris. Furthermore, reduced activity of capture reagent in the subsequent reprocessing is also observed. Sample and reagent mixing prior to equilibration with the capture support must be carried out prior to the reagents reaching the test device. This is frequently accomplished by using a long connecting tube which tends to result in contamination and requires considerable lengths of time for liquid to reach the reaction container.
Japanese Patent Application No. J58,223,758 published Dec. 26, 1983, filed in the name of Kokaietal, describes a flow-through system and circulatory reaction device which claims to overcome many of the disadvantages of flow-through systems. The reaction tube is an open tube, having an opening at both ends. A filter is used to provide a support for immobilization of antibodies or antigens in a nozzle. Positive pressure is used to force the material through the cell and to wash the solid phase capture reagents. The system is mechanically complex--requiring operation of numerous syringe pumps. The system is operated at positive pressure and therefore subject to emission or release of toxic and infectious aerosols. Furthermore, since the system makes use of a packed capture bed with fluid flow in one direction, diminished flow and increased back pressure will result from plugging.